The estrogen receptor (ER) is a drug target that has been exploited clinically by selective estrogen receptor modulators (SERMs) that antagonize estrogenic effects in breast tissue and act as agonists in bone. The proto- type of the SERM drug class, tamoxifen (TAM) has for 3 decades been the standard of care in breast cancer therapy, although agonist activity in the uterus increases risk of endometrial cancer. Conversely, the therapeutic potential of ERa agonists has not been explored or exploited. Up to 80% of breast cancers are ER+, in which estradiol (E2) fuels tumor growth. Of these, 30-50% are unresponsive to TAM or develop resistance, leaving few treatment options. Paradoxically, prior to the introduction of TAM, both E2 and the ER agonist diethylstilbestrol achieved success in the treatment of breast cancer, but with unacceptable side effects, including agonist activity in breast and uterus. A partial agonist at ERa without estrogenic actions in normal gynecological tissues, but with the ability to regress TAM-resistant cancers is a compelling concept, validated by us in 2 animal models with 2 small molecules. The assembled team at UI, Greg Thatcher, John Katzenellenbogen, Terry Moore, and Deb Tonetti has all assays and chemistry in place to achieve the proposed aims: Aim 1. A library of ~350 com- pounds consisting of 4 lead series will be completed and screened using a FRET assay for SRC3 co-activator binding to ERa and ER. The ER interaction with co-activator is seen as the minimal predictor of phenotype. Radioligand displacement for ER represents a secondary, confirmatory assay. Partial agonists at ERa with agonist/inactive responses at ER will be obtained and iteratively optimized using data from Aim 2 to provide SAR development for computer-aided design. Aim 2. Selected ERa agonists from FRET screening will be studied using reporter assays in endogenous ERa (MCF-7; T47D) and in ER (43) mammary epithelial cells and endometrial cells to reveal potency, and agonist/antagonist pharmacology. Partial agonists will be further studied using PCR measurement of ERE, AP-1, and Sp1-sensitive genes. The primary phenotypic assay is apoptosis of TAM-resistant MCF7-5C cells. Counterscreens are proliferation of parent MCF7-5C and T47D cell lines. Aim 3. Drug metabolism in liver microsomes, multiplex CYP-inhibition, and oral plasma bioavailability will be used to refine ERa partial agonists as chemical probes. Our extensive experience with SERM metabolism and use of oral delivery in all preliminary data gives confidence in this approach. DMPK studies will provide final selection for animal studies. Three different TAM-resistant xenograft models will be used to test chemical probes and to determine therapeutic index for tumor regression. Safety will be predicted by uterine growth and parallel study of growth of estrogen-dependent T47D xenografts. Supportive mechanism of action studies are limited to ERa localization and target selectivity proteomics. Completion of these Aims will provide chemical probes to study the potential of novel partial agonists and allosteric modulators of ER function, and provide lead compounds for future testing in patient-derived xenografts of PKCa-overexpressing tumors as a prelude to drug development.